Rational design-based engineering of a thermostable phytase by site-directed mutagenesis.

Abstract

Phytases are enzymes that hydrolysis phytic acid and makes mineral phosphorus available to animals. Phytases face relatively extreme heating during food processing, thus thermostability plays an important role in industrial applicability of this enzyme. Herein, we report the design of a thermostable phytase with favorable biochemical properties and high enzymatic activity using molecular dynamics and rational design-based molecular engineering. Based on the crystal structure of E. coli phytase, bioinformatics analysis and docking binding energy measurement, S392F mutant was introduced by site-directed mutagenesis in order to improve thermostability of phytase through strengthen the bounding interactions. Wilde type and Mutated constructs were expressed in E. coli BL 21. The WT and manipulated phytase were purified; their biochemical and kinetic was investigated. Results revealed that recombinant WT and mutant phytase have optimum temperature of 50°C with no significance change but optimum pH of WT and mutant was respectively 5 and 6 with a pH shift. Furthermore, S392F phytase catalytic efficiency values showed significant improve of 25.6%, compared with the WT. Analysis of the retained enzymatic activity at high temperatures, indicated that despite of phytase stability reduction at high temperatures but mutant phytase showed more stable behavior in compare with WT phytase, So that at 70°C showed twice thermo stability and at 80°C and 90°C display respectively 74% and 78.4% improvement of thermostability compared to the wild-type. In conclusion, our results implied that the designed phytase could be a potential candidate for phytase manipulation research and industrial applications with improved thermostability.